Background
In recent years, it has become increasingly evident that many non-protein-coding regions of the genome are transcribed [
1], giving rise to non-coding RNAs (ncRNA) that play crucial roles in normal biological processes and human diseases [
2]. Within the diverse group of ncRNA, small non-coding RNAs (sncRNAs) have emerged as potential important regulators of gene expression [
3]. These RNA molecules are highly complex in terms of structural diversity and function. They are typically 19–35 nucleotides (nt) in length, interact with Argonaute family proteins [
4‐
7], and include microRNAs (miRNAs), and PIWI-interacting RNAs (piRNAs). Precise control of miRNA expression is crucial for keeping cells in normal physiological states, and dysregulation of miRNAs may lead to oncogenesis [
8,
9]. The miRNA pathway is thought to be important for spermatogenesis [
10] and several miRNAs have been found to be exclusively or preferentially expressed in the testis [
11]. Moreover, the miRNAs miR-371-373, miR-302 and miR-146 have previously been shown to display a TGCT-specific expression pattern [
12‐
14], indicating a potential role for miRNAs in TGCT pathogenesis.
Unlike miRNA expression, piRNAs are mainly restricted to the male germline [
5,
15‐
17], but have recently also been found in small amounts in somatic tissues, including cancer [
18‐
21]. It has been reported that piRNAs tend to be generated in a clustered fashion from specific loci in the genome [
5,
16], and that they are mainly transcribed from regions containing transposons and other repetitive elements [
2,
22]. They are generated from long primary transcripts that are processed by unknown endonucleases into mature piRNAs [
22,
23]. Their roles in humans are still unclear, but studies in model organisms suggest a role in transposable element regulation and DNA methylation [
23‐
28]. It is thought that they form piRISC complexes with PIWI proteins and target mRNAs for cleavage or translational repression by binding to complementary sequences in target mRNAs [
29,
30].
Other classes of sncRNAs have also been identified, including tRNA-derived small RNAs, a class of small RNA generated by specific endonucleases from mature tRNAs in response to certain conditions, such as oxidative stress, heat shock, or nutrient deprivation [
31‐
33]. Previous studies have shown that tRNA-derived small RNAs can be divided into two main groups; small tRNA fragments (tRFs) (~20 nt in length) and tRNA halves (~30 nt in length) [
32]. Their mode of action is still unclear, but they may influence cell proliferation, probably through translational arrest [
32,
33]. Yamasaki and coworkers showed that stress induction in mammalian cells leads to formation of tRNA halves that inhibit translation [
33]. This inhibitory effect is specific for sequences derived from the 5′ end of mature tRNAs [
34]. Recently, Keam and co-workers showed that HIWI2, a human PIWI homolog, binds 5′ tRNA-derived small RNAs in a human breast cancer cell line, indicating crosstalk between the piRNA and tRNA pathways [
20]. Similar tRNA-derived small RNAs have also been detected in all stages of mouse spermatogenesis [
35].
Small ncRNA may play an important role in TGCT. TGCT develops from carcinoma
in situ (CIS; alias intratubular germ cell neoplasia) lesions that may arise
in utero from primordial germ cells (PGCs) [
36] and is the most common malignancy in young men in most western countries [
37,
38]. The etiology of TGCT is largely unknown, although genetic components and conditions during pregnancy play a role [
39‐
41]. In CIS cells, the demethylation machinery maintains the genome in a generally demethylated state, much like in undifferentiated PGCs [
42], indicating an epigenetic component in the development of testicular neoplasia. Recently, downregulation of the PIWI-piRNA pathway in human TGCTs was shown by Ferreira and coworkers [
43]. Downregulation was associated with loss of LINE-1 methylation. Combined, these findings indicate that epigenetic disruption is a hallmark for the development of testicular tumors, and that it is affected by the sncRNA expression.
Despite the emerging biological significance of sncRNAs, most studies thus far have been conducted in model organisms. Therefore, the abundance, diversity, origin, and function of human sncRNAs are still relatively unknown. Although several studies have been performed on the role of miRNAs in spermatogenesis and TGCT, little is known about the presence and molecular function of other classes of human testicular sncRNAs. This exploratory study elucidates the presence and expression levels of small RNA populations in normal testicular tissue and TGCT histological subtypes. We analyzed the distribution of sncRNAs across the human genome by small RNA sequencing on RNA isolated from a total of 37 human samples.
Discussion
During the last decade, several novel classes of sncRNAs have been identified. These molecules are highly complex, and their biogenesis, molecular function and regulation are still largely unknown [
47]. With this study we provide, to our knowledge, the first comprehensive characterization of the small RNA sequences in different histological subtypes of TGCT, and testicular carcinoma
in situ, as well as in normal testis samples. Overall, we show that the human testis is highly abundant in miRNAs, piRNAs and tRNA-derived small RNAs. More specifically, the small RNA population in our dataset showed characteristics of canonical piRNAs, with an overall genomic distribution closely resembling that of a previous study [
5], as well as a large overlap with piRNABank [
46]. In addition, we confirm the findings from other studies showing that human piRNAs, like piRNAs in other organisms, have a strong preference for 5′ U [
25,
48‐
50]. Together, these results strongly imply that a large proportion of the small RNAs, are indeed piRNAs. In the normal samples, we also observed a slightly elevated proportion of 10A in sequences 24–30 nt in length, indicating that some piRNAs may be generated through the ping-pong mechanism [
25,
48,
49].
Most piRNAs are globally downregulated or completely lost in all TGCT histological subtypes. This is not surprising, as tumor tissue has fewer differentiated germ cells, the main producer of piRNAs [
5,
15‐
17] compared to normal testis. Global downregulation of piRNAs in TGCT is supported by the findings of Ferreira and co-workers, describing diminished levels of PIWI proteins and piRNAs both in TGCT primary tumors and cultured transformed cells [
43]. Among the TGCT samples, we found 1) no histology-specific piRNA profiles, 2) no enrichment for 5′U and 10A, and 3) a lower read count for sequences 24–30 nt in length. Combined, these data indicate a lack of sequences generated through the piRNA pathway in TGCT. Some piRNAs are, however, detected in the cancer samples, shown by both the small RNA sequencing and qPCR validation, probably originating from a few normal cells within the tumors/biopsies.
TGCT develops from CIS lesions that may arise
in utero in PGCs or gonocytes [
36]. Although PGCs or gonocytes are not present in normal testis, we have considered CIS to represent a transitional state between normal testis tissue and TGCT, evident by the intermediate levels of 5′ U/10A enrichments observed in CIS samples (Fig.
1b). Only a proportion of the tubular cells will be affected in CIS (5-10 % abnormal cells [
51] in a 100 % CIS sample), generating a mix of normal spermatogenic and neoplastic cells. Almstrup and co-workers have previously determined the three CIS samples included in our study to contain 10 %, 50 %, and 100 % affected seminiferous tubuli, respectively [
51]. The samples do not cluster together in the piRNA dendrogram (Fig.
3a), probably due to the differences in the amount of neoplastic cells within the samples. Interestingly, the sample containing the highest amount of affected tubules cluster together with an embryonic carcinoma (EC) sample, whereas the sample containing the lowest amount of affected tubules clusters together with the healthy controls.
The high abundance of tRNA-derived RNAs 32–36 nt in length, representing tRNA halves, is in accordance with other studies [
20,
35]. Differential expression analyses of tRNA-derived sequences showed that despite their high abundance, no tRNA halves are differentially expressed between TGCT and normal tissue. This indicates that the population of tRNA halves is not affected by tumorigenesis. Most tRNA halves were found to be derived from the 5′ end of mature tRNA [
20,
35], suggesting that they are not degradation products, but rather processed tRNAs. The overrepresentation of only a few tRNAs, is similar to the results in other studies [
20,
35].
In addition to tRNA halves, we identified tRNA-derived sequences ~20 nt in length, corresponding to tRNA Fragments (tRFs) [
47]. These tRFs may be produced from the 5′- or 3′-end of mature tRNAs by Dicer, and associate with AGO proteins to participate in various processes of transcriptional and post-transcriptional regulation [
52‐
54]. Not much is known about the role of tRNA-derived small RNAs in cancer. It it has been reported that the levels of mature tRNAs are generally elevated in cancer [
55], whereas our results indicate that the levels of tRNA halves and tRFs are relatively stable in TGCT. The findings by Keam et al., showing that tRNA-derived small RNAs bind to HIWI2 [
20], indicate crosstalk between sncRNA pathways and may indicate that the sncRNA classes are more overlapping in terms of function and biogenesis than previously thought. Our results, however, show that the response to the carcinogenic process differs between these pathways, suggesting independent regulation of their biogenesis. More research is needed to elucidate the potential targets of tRNA-derived small RNAs and their role in cancer, including in TGCT.
Whereas piRNAs are almost exclusively found in the male gonad [
5,
16,
17], miRNAs are expressed in most cell types. The miRNA population varies, however between tissues. We speculate that the observed differences in miRNA profiles are driven by differences in the cellular origins of the TGCT subtypes, whereas piRNAs are lost in the carcinogenic cells due to the spermatogenesis-specific expression of this pathway. This lack of piRNA defense in CIS and TGCT cells may be a factor in testicular carcinogenesis, causing reduced ability to prevent chromatin instability. Diminished piRNA expression and hypomethylation events at LINE-1 loci in TGCT are supported by the findings of Ferreira et al. [
43] and Ushida et al. [
56].
Our results confirm previous findings indicating that miRNAs have a relevant role during testicular carcinogenesis, since overexpression of the miR-371-373, miR-302 and miR-367-3p clusters was noted in malignant TGCT tissue [
12,
57,
58]. These miRNAs are implicated in TGCT development [
13], maintenance of pluripotency [
59], and in cisplatin sensitivity [
60]. A study using a genetic screen of primary human cells supported this observation and found that both miR-372-373 and miR-302 may act as TGCT oncogenes through inhibition of target genes such as Large Tumor Suppressor homolog 2 (LATS2) [
12]. These miRNAs are all highly upregulated in TGCT, indicating a role as oncogenes in TGCT tumorigenesis. Also among the significantly differentially expressed miRNAs are miR-200c and miR-141, both belonging to the miR-200c/141 cluster, which has been found to act as inhibitors of the epithelial-to-mesenchymal transition, tumor cell invasion, and metastasis in several cancers [
61‐
63]. Counter indicative of their role as tumor suppressors, these miRNAs were also found to be upregulated in TGCT. However, there are some tumor types in which upregulation of the miR-200c/141 cluster has been observed, including ovarian carcinoma and endometrial carcinoma [
64,
65].
A limitation of the present study is the possible presence of non-piRNAs among the 25–32 nt long sequences. Although we enrich for phosphorylated small RNAs with a 5′-OH group in the library preparations, degraded RNAs and other non-phosphorylated RNAs may be co-sequenced. Compared to using PIWI protein immunoprecipitation [
5,
17], our method gives a broader range of small RNAs and a somewhat higher degree of non-targeted sequences. The strong preference for 5′U and the large overlap with piRNABank sequences, indicate that most of our sequences 25–32 nt in length are indeed piRNAs. In addition, differential expression analysis of only the sequences overlapping with piRNABank showed a similar expression pattern, indicating a true loss of the piRNA pathway in TGCT.
Another possible bias in the small RNA expression measurements is differences in RNA extraction protocols [
66]. Most normal samples were extracted using a phenol-free protocol, while the TGCT sample RNA extraction included a phenol step. To investigate this weakness in our design, the small RNA profiles of the available phenol-free (
n = 9) and phenol extraction (n = 3) of the normal samples were compared. No significant differential expression was found between samples prepared with the two extraction methods. Regardless, a replication study is needed to confirm potential biomarkers.
There is a need for more sensitive biomarkers for TGCT detection and surveillance [
67]. Several of the miR-371-373 and miR-302/367 cluster members have shown a sufficiently strong association with TGCT [
12,
57,
58] to serve as biomarkers of TGCT. Accordingly, serum levels of these miRNAs have been investigated and were found to be significantly higher in TGCT patients than in healthy controls, as well as display decreasing levels upon treatment [
68‐
70]. Among the miRNAs members from these clusters, miR-371a-3p and miR-367-3p have been considered most promising [
69,
71]. We have confirmed the association between TGCT and high expression of the miR-371-373 and miR-302/367 clusters. Despite our findings that differentially expressed piRNAs and tRNA-derived small RNAs are mostly downregulated in TGCT, they may have potential as biological markers. More research is needed to determine the role of these sequences in TGCT carcinogenesis and their potential clinical use.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Contributed equally: TBR and KF. Conceived and designed the experiments. All authors. Performed the statistical analyses: TBR. Analyzed the data: TBR, KF and EE. Contributed samples: TBH and RIS. Wrote the paper: TBR, KF and EE. Initiated the project and supervised the study: TG. All authors read and approved the final manuscript.